Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice

S. R. BORNSTEIN,*,1 T. TAJIMA,*,2 G. EISENHOFER,† A. HAIDAN,‡ AND G. AGUILERA**Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of ChildHealth and Human Development and †National Institute of Neurological Disorders and Stroke,National Institutes of Health, Bethesda, Maryland 20892-1862, USA; and ‡Department of InternalMedicine, University of Leipzig, Germany

ABSTRACT Deficiency of 21-hydroxylase (21-OH),one of the most common genetic defects in humans,causes low glucocorticoid and mineralocorticoid pro-duction by the adrenal cortex, but the effect of thisdisorder on the adrenomedullary system is unknown.Therefore, we analyzed the development, structure,and function of the adrenal medulla in 21-OH-deficient mice, an animal model resembling humancongenital adrenal hyperplasia. Chromaffin cells of21-OH-deficient mice exhibited ultrastructural fea-tures of neuronal transdifferentiation with reducedgranules, increased rough endoplasmic reticulumand small neurite outgrowth. Migration of chromaf-fin cells in the adrenal to form a central medulla wasimpaired. Expression of phenylethanolamine-N-methyltransferase (PNMT) was reduced to 27 6 9%(P<0.05), as determined by quantitative TaqManpolymerase chain reaction, and there was a signifi-cant reduction of cells staining positive for PNMT inthe adrenal medulla of the 21-OH-deficient mice.Adrenal contents of epinephrine were decreased to30 6 2% (P<0.01) whereas norepinephrine anddopamine levels were reduced to 57 6 4% (P<0.01)and 50 6 9% (P<0.05), respectively. 21-OH-defi-cient mice demonstrate severe adrenomedullary dys-function, with alterations in chromaffin cell migra-tion, development, structure, and catecholaminesynthesis. This hitherto unrecognized mechanismmay contribute to the frequent clinical, mental, andtherapeutic problems encountered in humans withthis genetic disease.—Bornstein, S. R., Tajima, T.,Eisenhofer, G., Haidan, A., Aguilera, G. Adrenomed-ullary function is severely impaired in 21-hydroxy-lase-deficient mice. FASEB J. 13, 1185–1194 (1999)

Key Words: 21-OH deficiency z adrenal medulla z catechol-amine synthesis

Congenital adrenal hyperplasia (CAH)3 due to21-hydroxylase (21-OH) deficiency is among themost common genetic disorders in humans (1). Thedefective or absent enzyme results in glucocorticoidand mineralocorticoid deficiency. Classical 21-OH

deficiency is a life-threatening disorder, causing se-vere salt wasting and eventually death if not diag-nosed and treated promptly (1, 2). The decreasedglucocorticoid production results in activation of thehypothalamic-pituitary-adrenal (HPA) axis with hy-persecretion of pituitary corticotrophin (ACTH),hyperplasia of the adrenal cortex, and overproduc-tion of precursor steroids, which are shunted intothe androgen biosynthetic pathway (3). This causessymptoms and signs of androgen excess includinghirsutism, acne, disproportionate advancement ofbone age, menstrual problems, diminished bonemass, infertility, and learning disabilities (1, 4–7).

Despite advances in our understanding of themolecular events causing congenital adrenal hyper-plasia, patients with this disorder continue to haveproblems reflecting inadequacy of the current ther-apeutic approach (2, 8–11). The complex patho-physiology of 21-OH deficiency is far from beingunderstood, and replacement therapy with glucocor-ticoids and mineralocorticoids often fails to normal-ize dysfunction of the HPA axis, growth, develop-ment, and well-being, suggesting alterations in othersystems (4, 5, 9, 12, 13).

The adrenocortical and adrenomedullary systemsare intimately linked both anatomically and func-tionally in the adrenal gland (14, 15). From in vitrostudies, it is well established that glucocorticoids arerequired for the survival and maintenance of chro-maffin cells and their ability to produce epinephrine(16, 17). The effect of impaired glucocorticoid se-

1 Correspondence: Developmental Endocrinology Branch,NICHD, NIH, Bldg. 10, Rm. 10n262, 10 Center Drive MSC1862, Bethesda, Maryland 20892-1862, USA. E-mail:Bornstes@mail.nih.gov

2 Department of Pediatrics, School of Medicine, HokkaidoUniversity, Sapporo Kita-ku N15 W7, Japan

3 Abbreviations: ACTH, corticotrophin; CAH, congenitaladrenal hyperplasia; HPA, hypothalamic-pituitary-adrenal;HPLC, high-performance liquid chromatography; 21-OH,21-hydroxylase; PCR, polymerase chain reaction; PNMT, phe-nylethanolamine-N-methyltransferase; RER, rough endoplas-mic reticulum; RT, reverse transcription; SIF, small intenselyfluorescent; TBS, Tris-buffered saline.

11850892-6638/99/0013-1185/$02.25 © FASEB

cretion on sympathoadrenomedullary function hasnever been studied in 21-OH deficiency or othertypes of CAH. In this study, the 21-OH-deficientmice, an animal model of CAH, were used to analyzethis problem. The mice that have a deletion of theCYP21 gene and impaired 21-OH activity are a modelfor the human classic 21-OH deficiency that occursin ;1:14,000 live births due to gene deletion or geneconversion (1, 13). As in human disease, the lack ofglucocorticoids results in adrenocortical hyperplasiaand accumulation of precursors (18). The majorityof affected mice die within a week if not treated withglucocorticoids and mineralocorticoids (19). Immu-nohistochemistry and electron microscopy were usedto analyze both adrenocortical and adrenomedullarystructures in this animal model. Adrenal catechol-amine levels were determined by liquid chromatog-raphy. The expression of mRNA for phenylethano-lamine-N-methyl transferase (PNMT), the enzymeresponsible for epinephrine production was quanti-tatively assessed by TaqMan polymerase chain reac-tion (PCR).

MATERIALS AND METHODS

Animals and experimental protocols

Heterozygous 21-deficient mice (H-2 aw18 haplotype), kindlyprovided by Dr. Toshihiro Shiroishi, Institute of Genetics,Shizuoka-ken, Japan, and wild-type C5BL10J mice purchasedfrom Jackson Labs (Bar Harbor, Maine) were maintainedaccording to NIH guidelines with a 12 h light-dark cycle andfree access to food and water. The presence of a vaginal plugon the morning after mating was set as day 0.5 of gestation. Asdescribed previously, the gestation period in heterozygousmice was 1–2 days longer than that expected in wild-type mice(17, 18). All dams were treated with 5 mg of dexamethasone/day subcutaneous (s.c.) from gestational day 20 until deliveryin order to prevent death of homozygous pups at birth. Pupswere treated with corticosterone (5 mg/day) and fludrocorti-sone (0.025 mg/day) s.c. until day 14, followed by corticoste-rone in the drinking water until day 21. Animals wereanalyzed at 1 wk and 1 month of age. Each animal was killedwithin 3 min of being removed from its home cage and theprocedures were performed sequentially in a separate room.All animal protocols were approved by the Animal Users CareCommittee, NICHD, NIH.

Determination of genotype and 21-OH deficiency

Genomic DNA was extracted from livers or tails using stan-dard procedures described previously (17). A 950 bp cDNAfragment encoding exons 3 to 9 of the mouse CYP21 cDNAwas prepared by PCR using 500 ng of total adrenal RNA(prepared with TRIzol reagent, Life Technologies, Gaithers-burg, Md.) and the GeneAmpRNA PCR kit (Perkin Elmer,Foster City, Calif.). The upstream primer was 59-GAAAGAT-GGACTTGGACCTGTCCT-39 and the downstream primerwas 59-AGGGTAGTCATAGCCGGAGAT-39. PCR was per-formed using 500 ng of mouse adrenal RNA as a templateunder the following conditions: 30 cycles, 1 min at 94°C; 1min at 58°C; and 3 min extension at 72°C. The blunt-ended

PCR product was cloned with the use of a TA cloning kit(InVitrogen, Carlsbad, Calif.) and used to prepare randomprimer radiolabeled probes for Southern blot analysis ofBgl-II digests of the genomic DNA (17). As previously shown,wild-type mice showed two bands corresponding to the activeCYP21-B and the functionally inactive CYP21-A genes. Ho-mozygous mice allowed a single smaller molecular bandcontaining CYP21-A, whereas heterozygotes showed the threebands.

Electron microscopy

Adrenal glands were removed, dissected, and fixed for 3 h in2% formaldehyde and 2% glutaraldehyde in 0.1 M phosphatebuffer, pH 7.3. Tissue slices were postfixed for 90 min (2%OsO4 in 0.1 M cacodylate buffer pH 7.3), dehydrated inethanol, and embedded in epoxy resin. Ultrathin sectionswere stained with uranyl acetate and lead citrate and exam-ined at 80 kV in a Philips EM 301.

Immunohistochemistry

For specific staining of chromaffin cells, paraffin sections ofadrenals were immunostained using a mouse anti-tyrosinehydroxylase antibody (Boehringer, Mannheim, Germany).Immunohistochemistry for PNMT was performed using amonoclonal rabbit anti-mouse antibody (courtesy of C. Gro-the, Freiburg, Germany). Sections were deparaffinized andpretreated in the microwave in 10 mM citrate buffer pH 6.0for 3 3 5 min (PNMT) or in Tris-buffered saline, pH 7.6(TBS) containing 0.5% Triton X-100 for 2 min (tyrosinehydroxylase). Endogenous peroxidase activity was quenchedby incubation in 0.5% hydrogen peroxide in TBS containing10% methanol for 20 min. Immunostaining was performed bythe avidin-biotin technique using the UniTect immunohisto-chemistry detection system (Dianova, Hamburg, Germany).After preincubation with 2% normal horse serum for 30 min,sections were incubated with the anti-tyrosine hydroxylaseantibody diluted 1:10 in TBS containing 2% normal horseserum at 4°C overnight. For immunostaining of PNMT,nonspecific binding was blocked by 3% normal goat serumfor 45 min. Subsequently, sections were incubated with thePNMT-antibody at 4°C overnight. The antibody was diluted1:250 in Dako Antibody Diluent (Dako, Hamburg, Germany)containing 1% goat serum and 0.3% Triton X-100. Afterrinsing in TBS, sections were incubated with biotinylated linkantibodies for 30 min and avidin-biotin-peroxidase complexfor 30 min. Visualization of the immune complex wasachieved by incubating the sections in AEC (3-amino-9-ethyl-carbazole) chromogen System (Dianova) for 15 min andcounterstaining with hematoxylin.

TaqMan PCR

For quantitation of PNMT mRNA expression, we applied thenovel technique of Real Time Quantitative PCR (TaqManPCR) (20) using the 7700 Sequence Detector (Perkin ElmerApplied Biosystems). The amount of PNMT mRNA wasmeasured using adrenocortical RNA and the following prim-ers and TaqMan-probe designed from the mouse PNMT genesequence (GenBank L12687) by Primer Express (PE): for-ward 59-GTC GGG ACG GGT TCT CAT T-39; reverse 59-CCAAGA AGT CTG TCA TGG TGA TG-39; TaqMan-probe 59(FAM)-CTC CGG CCC CAC CAT ATA TCA GCT G-(TAMRA)39. 18S RNA levels were detected with the TaqMan ribosomalRNA control reagents (PE).

Total RNA was isolated from the adrenals of wild-type and21-OH-deficient mice using the RNAeasy kit from QIAGEN

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(Valencia, Calif.). Traces of DNA were removed by digestionwith RNase free DNase A (Boehringer, Indianapolis, Ind.) Aone-step reverse transcription-PCR (RT-PCR) was performedaccording to the protocol supplied with the TaqMan GoldRT-PCR kit (PE). Reactions contained 1 3 TaqMan buffer A,5.5 mM MgCl2, 0.3 mM each dATP, dCTP, and dGTP, 0.6 mMdUTP, 0.4 U/ml RNase inhibitor, 0.025 U/ml AmpliTaq GoldDNA polymerase, and 0.25 U/ml MultiScribe reverse tran-scriptase. Primers and probes were added at the followingconcentrations: 900 nM for the PNMT forward and reverseprimer, 200 nM for the PNMT TaqMan probe, and 50 nM for18 S primers and probe. After reverse transcription at 49°Cfor 30 min, AmpliTaq Gold was activated at 95°C for 10 min.Thermal cycling proceeded with 40 cycles of 95°C for 15 s and60°C for 1 min. Input RNA amounts were calculated withrelative standard curves for both PNMT and 18S. The amountof PNMT mRNA was corrected by division by the amount of18S RNA in each sample.

Catecholamine measurements

Tissues were homogenized in 5 volumes of ice-cold 0.4 Mperchloric acid containing 0.5 nM EDTA. Homogenates werecentrifuged at 3000 rpm at 4°C, and the supernatants werestored in aliquots until assayed. Catecholamines in superna-tants were determined by liquid chromatography with elec-trochemical detection after a batch alumina extraction, asdescribed previously in detail (21).

Plasma steroid levels

Blood was collected from newborn mice by decapitation at9:00 am using nonheparinized capillary tubes. Plasma corti-costerone levels were determined quantitatively by high-performance liquid chromatography (HPLC) as describedpreviously (22). Plasma progesterone concentrations weredetermined using commercial kit reagents from DiagnosticSystems Laboratories, Inc. (Webster, Tex.). Controls wereused in the low and high section of the standard curve. Theinter- and intra-assay coefficients of variation were both , 5%.

Statistical analysis

Data are presented as mean 6 se and were analyzed byStudent’s t test or the Mann-Whitney-U test, depending on thedistribution pattern of the data.

RESULTS

Adrenal morphology

Mice deficient in 21-OH showed marked changes inadrenal structure compared with wild-type mice (Fig.1A, B). The adrenal cortex of mutant mice wasmarkedly enlarged with hyperplasia of adrenocorti-cal cells. There was no normal zona glomerulosa andfasciculata-like cells extended to the capsule. Themigration of chromaffin cells in the center of thegland was incomplete, with single cells and islets ofchromaffin cells remaining within the adrenal cortexas demonstrated by the presence of tyrosine hydrox-ylase or PNMT (not shown) stained cells (Fig. 1C). Inaddition, chromaffin cells interspersed within the

cortical tissue formed thin cellular extensions. Thesealterations were present in both 1-wk-old and1-month-old mice.

Electron microscopy

At the ultrastructural level, adrenocortical cells ofwild-type mice demonstrated normal smooth endo-plasmic reticulum, liposomes, and the characteristicmitochondrial structure, elongated with tubulola-mellar cristae in glomerulosa cells and round withtubulovesicular cristae in fasciculata/reticularis cells.Adrenocortical cells of 21-OH-deficient mice showedenlarged mitochondria with scarce internal mem-branes, frequently demonstrating myelin-type figuresand lipidic inclusions.

Chromaffin cells of wild-type animals had thecharacteristic ultrastructural feature of neuroendo-crine cells with an ample presence of membrane-bound, secretory granules so-called dense-core vesi-cles ;60 to 400 nm in greatest dimension. (Fig. 2A,C) Two principal types of granule-containing cellswere found in the normal adrenal medulla: 1) epi-nephrine containing large, round or elongated me-dium-density granules with a particulate substruc-ture; and 2) small norepinephrine containingelectron-dense granules within a large lucent vacu-ole. (Fig. 2C) In the 21-OH-deficient mice, there wasa conspicuous depletion of secretory vesicles. (Fig.2B) The remaining granules were predominantlyelectron-dense norepinephrine granules within alarge lucent vacuole (Fig. 2D). Chromaffin cells werefrequently in contact with adrenocortical cells andformed small neurite-like outgrowths. Cells con-tained large areas of rough endoplasmic reticulum(RER). Frequently, the RER was dilated and vesicu-lated.

PNMT expression

PNMT mRNA expression in homozygous 21-OH-deficient mice was significantly reduced to 27 6 9%(P,0.05) compared with wild-type mice, as shown byTaqMan PCR. (Fig. 3) Concomitantly, the numberof chromaffin cells staining for PNMT was markedlydecreased in the 21-OH-deficient mice. (Fig. 4A, B).There was no remarkable change in the number ofchromaffin cells staining for tyrosine hydroxylase in21-OH-deficient mice compared with wild-type mice.

Adrenal catecholamine levels

Consistent with the altered chromaffin cell structure,adrenal catecholamine levels were significantly re-duced in 21-OH-deficient mice. Dopamine levels inthe 21-OH-deficient mice were significantly reducedto 50 6 9% (P,0.05) and norepinephrine levels to

118721-OH DEFICIENCY AND ADRENOMEDULLARY FUNCTION

Figure 1. Morphological changes of adrenals of adult 21-OH-deficient mice compared with wild-type animals. A) Wild-type adrenalsdemonstrate a normal zonation with a capsule (CAP), outer zona glomerulosa (ZG), zona fasciculata (ZF); zona reticularis (ZR), andan inner zona medullaris (ZM) (3200). B) The adrenal cortex of the 21-OH-deficient mice is hyperplastic with enlargedadrenocortical cells (small arrows). There is no regular zonation, with fasciculata-like cells reaching to the outer capsule (largearrows). Islets and single chromaffin cells identified by immunostaining for tyrosine hydroxylase are located within the cortex. C)Cortical cells and chromaffin tissue are intermingled and form direct cellular contact (large arrows) (3200). Compared to wild-typeanimals in 21-OH-deficient mice, chromaffin cells show an irregular staining pattern for tyrosine hydroxylase. Some cells form tyrosinehydroxylase positive cellular extensions representing outgrowth of small neurites (small arrows).

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57 6 4% (P,0.01) compared with the levels inwild-type animals. (Fig 5A, B). Epinephrine levels inhomozygous 21-OH-deficient mice were 1258 6 92ng/adrenal compared with 4227 6 529 ng/adrenalin controls (P,0.01), representing a reduction to30 6 2% of the levels in the controls. (Fig. 5C).

Plasma steroid levels

Serum corticosterone levels as determined by HPLCwere significantly reduced in 21-OH-deficient micecompared with wild-type mice (6.5861.1 ng/ml vs.

44.466.2 ng/ml). Plasma progesterone levels weremarkedly elevated in 21-OH-deficient mice com-pared with wild-type mice (0.960.1 ng/ml vs.269.2632.4 ng/ml).

DISCUSSION

The findings of our study indicate that glucocorti-coid insufficiency in 21-OH-deficient mice causesmajor structural changes in the adrenomedullarysystem and impairment of adrenal catecholamineproduction.

Figure 2. Electronmicrograph of chromaffin cells of 1-wk-old 21-OH-deficient mice as compared with wild-type animals stainedwith uranyl acetate and lead citrate. A) On the ultrastructural level, the cytoplasm of chromaffin cells of controls is filled withdensely grouped catecholamine storing secretory vesicles 50 to 450 nm in size. NUC, nucleus; bar 5 0.5 mM. B) In the21-OH-deficient mice, there is a conspicuous reduction in number of secretory granules. Remaining granules are frequentlyfound lining up at the cell membrane (arrowhead). Cells contain a large amount of rough endoplasmic reticulum (RER) withdilated cisternae. The RER is frequently found in the perinuclear area. A shrunken nucleus with condensed chromatin as anearly sign of programmed cell death can be seen (arrows). Chromaffin cells are in direct contact with adrenocortical cells.Adrenocortical cells demonstrate liposomes (LIP) and large round mitochondria with sparse tubulovesicular internalmembranes (MIT) (bar 5 0.5 mM). C) In wild-type animals, the majority of cells contain secretory vesicles with round orelongated medium-density granules with a granular substructure representing epinephrine-storing-vesicles (arrows). D) In21-OH-deficient mice, the remaining granules are predominantly electron-dense, norepinephrine-containing vesicles lying inlarge lucent vacuoles (small arrows). The empty vacuoles endowed with ribosomes are not secretory granules but vesiculatedRER (large arrows). MIT, mitochondria; bar 5 0.1 mM.

118921-OH DEFICIENCY AND ADRENOMEDULLARY FUNCTION

There was a fourfold reduction in adrenal epi-nephrine levels and a twofold decrease in norepi-nephrine levels. This finding was consistent with asignificant reduction in PNMT mRNA and PNMTprotein as well as epinephrine-secreting vesicles inthe adrenal medullae of 21-OH-deficient mice. Inlate embryonic, neonatal, and adult chromaffin cells,PNMT expression is regulated by glucocorticoids(16, 17). In vitro, glucocorticoids have been shown toinduce the enzyme PNMT, which is necessary for theproduction of epinephrine in medullary cells (23–25). Epinephrine deficiency has been reported inhypocorticotropic hypopituitary children (26).Therefore, the absence of normal adrenocorticalcorticosterone secretion is likely to be responsiblefor an impaired production of adrenal cat-echolamines in 21-OH-deficient mice. Clearly, othercorticosterone precursors that are overproduced inthese animals cannot compensate for the loss ofcorticosterone. Also, exogenous replacement of syn-thetic corticosteroids in the 7-day-old mice did notrestore chromaffin cell function. This is in line withreduced epinephrine levels in patients with adreno-cortical insufficiency due to Addison’s disease thatare adequately replaced with hydrocortisone (27).The decreased levels of dopamine, norepinephrine,and epinephrine indicate that reduction of epineph-rine was not due simply to reduced PNMT, butprobably also involved decreased tyrosine hydroxyla-tion. This agrees with previous reports, demonstrat-ing that glucocorticoids are also involved in theinduction of tyrosine hydroxylase (28–30). Never-theless, the predominant decrease in epinephrinecompared with norepinephrine and dopamine areconsistent with the major effect on PNMT. Thissuggests that circulating glucocorticoid levels are not

sufficient to maintain normal adrenomedullary func-tion, and the local influence of very high glucocor-ticoid levels directly from the adrenal cortex isnecessary for a normal chromaffin cell function.Thus, local intra-adrenal levels of glucocorticoidsafter replacement therapy are not sufficient to nor-malize the function of the adrenal medulla.

In addition to the severely impaired adrenal cate-cholamine production, the adrenals of the 21-OH-deficient mice demonstrated major structural changes.As sympathetic principal neurons, adrenomedullarychromaffin cells originate from neural crest precursorcells and migrate into the adrenal ‘anlagen’ where theylater differentiate into chromaffin cells in the adrenalmedulla under the influence of adrenocortical steroids(17, 31). A third cell type, small intensely fluorescent(SIF) cells, with intermediate characteristics betweenneurons and chromaffin cells have been described inculture studies (32–34). While converting to neurons,adrenal chromaffin cells transiently assume an inter-mediate phenotype resembling SIF cells, underscoringthe remarkable plasticity of chromaffin cells and theimportance of environmental factors in neural crestdevelopment. It is interesting that the phenotype of thechromaffin cells in the 21-OH-deficient mice resem-bled the SIF cells, with formation of small neurites,reduction of catecholamine-storing vesicles, and amplerough endoplasmic reticulum. Furthermore, in con-trast to the central location of normal adrenal medul-lae in wild-type mice, chromaffin cells in the adrenalsfrom 21-OH-deficient mice were intermingled with thehyperplastic cortical tissue, suggesting a defect in themigration process.

Does this finding in 21-OH-deficient mice relate tothe situation in human with CAH? In contrast to the21-OH-deficient mice, which have very low cortico-sterone levels, the adrenals of CAH patients cansynthesize a baseline amount of cortisol due toincreased ACTH secretion and adrenal hyperplasia.However, once the CAH patients receive adequatereplacement therapy with exogenous glucocorti-coids, the suppression of plasma ACTH levels and ofadrenal hyperplasia results in extremely low endog-enous intra-adrenal cortisol production (,1 mg/dl)(35). In a preliminary study, we found significantlydecreased 24 h urinary epinephrine concentrationsin patients with classical 21-OH deficiency uponadequate steroid replacement therapy (S. R. Born-stein, G. Eisenhofer, and D. Merke, unpublishedobservation). This strongly suggests that the adreno-medullary impairment described in 21-OH-deficientmice also occurs in humans with this commongenetic disorder.

The defect in adrenomedullary function may haveseveral implications. Clearly, the two endocrine systemswithin the adrenal form a functional unit, and analteration in one system will certainly affect the other

Figure 3. Quantitative PNMT mRNA in the adrenals ofwild-type animals and of 21-OH-deficient mice as determinedby TaqMan PCR. Amount of PNMT RNA is expressed aspercent of reduction of wild-type levels (n54, or P,0.05).

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Figure 4. Immunoreactive PNMT in the adrenal medulla of 1-month-old wild-type (A) and homozygous 21-OH-deficient mice(B). The number of cells staining positive for PNMT is markedly reduced in the 21-OH-deficient mice.

119121-OH DEFICIENCY AND ADRENOMEDULLARY FUNCTION

(15). It has recently been demonstrated that chromaf-fin cells are in close cellular contact with adrenocorticalcells (14, 36–38), immune cells (39, 40), nerve cells(41), and endothelial cells (42) in normal adrenals.These paracrine intra-adrenal interactions within dif-

ferent components of the adrenal are important forbasal hormone production and play a role in thedevelopment, cell proliferation, circadian rhythm andzonation of the adrenal gland (15, 43–56). Therefore,it is possible that adrenomedullary insufficiency inCAH also influences adrenocortical differentiation andthat impaired adrenomedullary function due to glu-cocorticoid deficiency may contribute to the lack ofzonation found in these animals.

Second, an ACTH-independent, neuroadrenocor-tical regulation mediated through the sympathoad-renal system has been demonstrated to be importantin acute stress, fine-tuning of the adrenals, earlydevelopment, and chronic adaptation to stress ininflammation, sepsis, and mental disorders (15, 43,44, 57–60). Therefore, a defect in the neuroadreno-cortical regulation will contribute to an impairedadaptation to stress in patients with 21-OH defi-ciency. This may be particularly important duringearly infancy, when the adrenal neocortex has toadapt to extrauterine life after disruption of thefetoplacental unit by adjusting glucocorticoid levels(61).

Third, adrenomedullary dysfunction will contrib-ute to the hypoglycemia that is a common problemin CAH patients, and a defective adrenal medullacan also aggravate the blood pressure problems inpatients with congenital adrenal hyperplasia (62,63).

Finally, similar to the adrenal medulla, glucocorti-coid deficiency may also affect epinephrine synthesisin the brain (16, 64, 65). Epinephrine has beenreported to play an important role in tonic regula-tion of arousal, reward, and sensitivity to environ-mental stimuli and subjective well-being (66, 67). It isa critical factor in mental task performance andattention-deficit hyperactivity disorder (68–70).Therefore, it is possible that a defect in epinephrineproduction is responsible for the high frequency oflanguage/learning disabilities in children with con-genital adrenal hyperplasia (5, 71).

Conventional treatment with glucocorticoid andmineralocorticoid replacement cannot restore thealteration in the HPA axis and the sympathoad-renomedullary system. Note that recent studies inour laboratory have shown that adenovirus-mediatedtransfer of the human CYP21 gene to 21-OH-defi-cient mice successfully restore corticosterone forma-tion in the adrenal. Concomitant with the increase inglucocorticoid synthesis, we could demonstrate thatadrenal zonation, including formation of a normalmedulla and PNMT expression, could be partiallynormalized (unpublished results.)

In conclusion, our data demonstrate that glu-cocorticoid and mineralocorticoid deficiency as aresult of the 21-OH defect not only affects the HPAaxis and the renin-angiotensin-aldosterone system,

Figure 5. Adrenal catecholamine levels in wild-type andhomozygous 21-OH-deficient mice. Dopamine (A), norepi-nephrine (B), and epinephrine (C) levels were determined byliquid chromatography. Data are expressed as mean 6 se(bars); catecholamine production: ng/adrenal or mM/adre-nal; n 5 4 in each group (*P,0.05, **P,0.01).

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but also severely compromises the adrenomedullarysystem. This is important for a better definition ofsymptoms and therapeutic strategies in 21-OH defi-ciency and should be considered in the clinicalmanagement of this common genetic disorder.

Supported in part by grants from Deutsche Forschungsge-meinschaft [EH 161/2–4] and by a Heisenberg grant, both toDr. S.R.B. We would like to thank D. Merke, U. Lopatin, andM. Connors for helpful comments.

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Received for publication November 4, 1998.Revised for publication December 12, 1998.

1194 Vol. 13 July 1999 BORNSTEIN ET AL.The FASEB Journal